Our overall objective continues to be to define the critical molecular mechanisms culminating in lethal liver cell injury. However, we have expanded the scope of our program from focusing entirely on cell death by necrosis to now addressing apoptosis as well. Based on our work, we have generated the novel general hypothesis that the mechanism by which a cell dies (necrosis vs. apoptosis) is determined by the activation and regulation of key proteases within specific cellular compartments. Specifically, our preliminary data suggest enhanced calpain activity causes cell necrosis by inducing the mitochondrial membrane permeability transition while a stimulation of nuclear serine protease activity promotes cell death by apoptosis. We will now employ current and complimentary molecular, biochemical and cell biological techniques to ascertain the regulatory and mechanistic aspects of calpain and serine protease activity during lethal liver cell injury by necrosis and apoptosis, respectively. Our proposal has two specific aims. FIRST, we will determine the mechanistic role of calpain protease activity in necrosis by directly testing the hypothesis that: a) the susceptibility of liver cell subtypes to necrosis is determined by quantitative expression of calpain mRNA, protein and catalytic activity; b) pathophysiologic insults stimulate calpain protease activity by causing increases of cytosolic free Ca++ (Ca++i) and/or enhanced phospholipid degradation; and c) calpain protease activity causes the mitochondrial membrane permeability transition. SECOND, we will determine the identity, regulation, and mechanistic role of protease activity in apoptosis by directly testing the hypothesis that: a) enhanced nuclear serine protease activity occurs in apoptosis and precedes DNA cleavage; b) proteolysis during apoptosis is regulated by intracellular divalent cations and pH; and c) proteolysis causes apoptosis by degrading nuclear matrix proteins. The studies will employ cultures and/or suspensions of isolated rat liver hepatocytes, cholangiocytes, and rat hepatoma cell lines. In addition, we have developed and validated novel fluorescent approaches to quantitate protease activity in single cells as well as cell fractionation techniques to obtain highly purified nuclei and mitochondria. We are now uniquely poised to pursue our long-term goals which are to provide new information regarding proteolysis as a mechanism of lethal injury of liver cells during clinically relevant models of cell injury. Successful completion of this proposal may identify treatment modalities (e.g., protease inhibitors) effective in preventing liver cell injury.